Epithelial sodium channels (ENaCs) mediate reabsorptive sodium transport across epithelial cell layers in conjunction with a basolateral Na+/K+-ATPase. These channels have a key role in the regulation of urinary Na+ reabsorption, extracellular fluid volume homeostasis and blood pressure. ENaC-dependent Na+ reabsorption across airway epithelia has a significant role in regulating the volume of airway surface liquids and mucociliary clearance. Important functional domains have been identified, including sites that affect channel gating, trafficking and cation selectivity. Studies proposed in this application will address questions regarding ENaC structure-function relationships. We recently identified sites within alpha- and gammasubunit extracellular domains where mutations affect channel gating kinetics and alter channel regulation by external Ni2+, Zn2+ and Na+ (Na+ self-inhibition). Experiments proposed in Aim 1 will use site-directed mutagenesis to explore the role of residues within these extracellular sites and the role of highly conserved cysteine residues in modulating channel activity in response to extracellular factors. Experiments proposed in Aim 2 will examine the structural organization of the channel's selectivity filter. Experiments proposed in Aim 3 will characterize a region within the carboxyl-terminus of the alpha subunit that is distinct from the PY motif and regulates ENaC trafficking. Successful completion of the studies proposed in this application will provide a more detailed understanding of key structural features of epithelial sodium channels.